Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2000-04-07
2001-10-30
Houtteman, Scott W. (Department: 1656)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S006120
Reexamination Certificate
active
06310198
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to the field of chemical synthesis of oligonucleotides. More particularly, the invention relates to the synthesis of extremely high purity oligonucleotides.
2. Summary of the Related Art
Since the discovery by Zamecnik and Stephenson (
Proc. Nati. Acad. Sci
. 75, 280 (1978)) that synthetic oligonucleotides can inhibit Rous sarcoma virus replication, there has been great interest in the use of oligonucleotides and oligonucleotide analogs having modified internucleotide linkages to control gene regulation and to treat pathological conditions. There have been many reports of successful use of antisense oligonucleotides to inhibit gene expression both in vitro and in vivo, either directly by binding to double stranded DNA, or, primarily, indirectly by inhibiting translation of mRNA.
Many reports of successful antisense inhibition of nucleic acid expression in vitro have been reported. For example, Rapaport and Zamecnik (U.S. Pat. No. 5,616,564) disclosed successful antisense inhibition of malaria in parisitized erythrocytes. See also Barker et al. (
Proc. Natl. Acad. Sci. USA
93, 514 (1996)). Oligodeoxyribonucleotide phosphorothioates have been found to inhibit immunodeficiency virus (Agrawal et al.,
Proc. Natl. Acad. Sci. USA
85, 7079 (1988); Agrawal et al.,
Proc. Natl. Acad. Sci. USA
86, 7790 (1989); Agrawal et al., in
Advanced Drug Delivery Reviews
6, 251 (R. Juliano, Ed., Elsevier, Amsterdam, 1991); Agrawal et al. in
Prospects for Antisense Nucleic Acid Therapy of Cancer and AIDS
, 143 (E. Wickstrom, Ed., Wiley/Liss, New York, 1991); and Zamecnik and Agrawal in
Annual Review of AIDS Research
, 301 (Koff et al., Eds., Dekker, New York, 1991)), and influenza virus (Letter et al.,
Proc. Natl. Acad. Sci. USA
87, 3420-3434 (1990)) in tissue culture. In addition, oligodeoxyribonucleotide phosphorothioates have been the focus of a wide variety of basic research (e.g., Agrawal et al.,
Proc. Natl. Acad. Sci. USA
87, 1401 (1990) and Eckstein and Gish,
Trends Biochem. Sci
. 14, 97 (1989)), enzyme inhibition studies (Mujumdar et al.,
Biochemistry
28, 1340 (1989)), regulation of oncogene expression (Reed et al.,
Cancer Res
. 50, 6565 (1990)) and IL-1 expression (Manson et al.,
Lymphokine Res
. 9, 35 (1990)) in tissue culture. A number of review articles report the many published studies of successful antisense inhibition in vitro. E.g., Uhlmann and Peyman,
Chem. Rev
. 90, 543 (1990).
A number of published reports disclose the successful antisense inhibition of nucleic acid expression in vivo. For example, Offensperger et al. (
EMBO J
. 12, 1257 (1993)) demonstrated in vivo inhibition of duck hepatitis B virus. Nesterova and Cho-Chung (
Nat. Med
. 1, 528 (1995)) demonstrated inhibition of tumor growth by a single subcutaneous injection of antisense phosphorothioate oligonucleotide targeted to the RI
&agr;
subunit of protein kinase A in nude mice. Several general reviews of in vivo antisense inhibition have appeared that discuss these and other studies demonstrating successful in vivo antisense inhibition of nucleic acid expression as well as applications for therapeutic use. See, e.g., Agrawal,
TIBTECH
14, 376 (1996); Field and Goodchild,
J. Exp. Opin. Invest. Drugs
4, 799 (1995).
These and other studies have proven sufficiently successful to justify extension to humans. A number of human clinical trials are currently ongoing, testing antisense oligonucleotides against a variety of disease causing targets, including HIV, CMV retinitis, ICAM, PKC, c-myb, and c-raf.
A necessary precursor to using antisense oligonucleotides to inhibit nucleic acid expression is the synthesis of the oligonucleotides. Various methods have been developed for the synthesis of oligonucleotides for such purposes. Early synthetic approaches included phosphodiester and phosphotriester chemistries. Khorana et al. (
J. Molec. Biol
. 72, 209 (1972)) discloses phosphodiester chemistry for oligonucleotide synthesis. Reese (
Tetrahedron Lett
. 34, 3143 (1978)) discloses phosphotriester chemistry for synthesis of oligonucleotides and polynucleotides. These early approaches have largely given way to the more efficient phosphoramidite and H-phosphonate approaches to synthesis. Beaucage and Caruthers (
Tetrahedron Lett
. 22, 1859 (1981)) discloses the use of deoxynucleoside phosphoramidites in polynucleotide synthesis. Agrawal and Zamecnik (U.S. Pat. No. 5,149,798) discloses optimized synthesis of oligonucleotides by the H-phosphonate approach.
Both of these modern approaches have been used to synthesize oligonucleotides having a variety of modified intemucleotide linkages. Agrawal and Goodchild (
Tetrahedron Lett
. 28, 3539 (1987)) teaches synthesis of oligonucleotide methylphosphonates using phosphoramidite chemistry. Connolly et al. (
Biochemistry
23, 3443 (1984)) discloses synthesis of oligonucleotide phosphorothioates using phosphoramidite chemistry. Jager et al. (
Biochemistry
27, 7237 (1988)) discloses synthesis of oligonucleotide phosphoramidates using phosphoramidite chemistry. Agrawal et al. (
Proc. Natl. Acad. Sci. USA
85, 7079 (1988)) discloses synthesis of oligonucleotide phosphoramidates and phosphorothioates using H-phosphonate chemistry.
A number of treatises and review articles have appeared that discuss the various synthetic approaches. E.g.,
Methods in Molecular Biology
, Vol. 20
, Protocols for Oligonucleotides and Analogs
, p. 63-80 (S. Agrawal, Ed., Humana Press 1993);
Methods in Molecular Biology, Vol
. 26
: Protocolsfor Oligonucleotide Conjugates
(Agrawal, Ed., Humana Press, Totowa, N.J. 1994);
Oligonucleotides and Analogues: A Practical Approach
pp. 155-183 (Eckstein, Ed., IRL Press, Oxford 1991);
Antisense Res. and Applns
. pp. 375 (Crooke and Lebleu, Eds., CRC Press, Boca Raton, Fla. 1993);
Gene Regulation: Biology of Antisense RNA and DNA
(Erickson and Izant, eds., Raven Press, New York, 1992).
Both phosphoramidite and H-phosphonate chemical syntheses are carried out on a solid support that is stored in a reaction vessel. The required reaction steps for coupling each nucleotide are detritylation, coupling, capping, and oxidation. For small scale (up to 1 &mgr;mole) synthesis, the total time for the addition of one nucleotide is about 6 minutes. An oligonucleotide, 30-mer in length, can be assembled in 180 minutes. Under these conditions, synthesized oligonucleotides are chemically pure and biologically active. However, when oligonucleotides are synthesized on a larger scale (up to 1 mmole), the time for addition of each nucleotide onto CPG is in the range of 30 to 60 minutes, requiring approximately 12-25 hours for assembling a 25-mer oligonucleotide. The increase in time is due to the volume of the solid support being used in synthesis. This increase in cycle time exposes the already assembled oligonucleotide sequence to all reaction steps (including dichloroacetic acid detritylation step, coupling step, oxidation step and capping step) for a longer time. This increase in total assembly time affects the yield as well as chemical and biological properties of the compound. The chemical and biological properties are mainly affected by depurination, base modifications, and the like.
To reduce the effects of these problems, it is possible to synthesize oligonucleotides using dimeric or multimeric synthons, thereby reducing the number of cycles, and thus the time required for synthesizing oligonucleotides. To this end, several investigators have worked toward developing acceptable dimeric or multimeric synthon approaches. Khorana (
Science
203, 614 (1979)) introduced the concept of multimeric synthons, using a phosphodiester approach. Crea and Itakura (
Proc. Natl. Acad. Sci. USA
75, 5765 (1978)), Reese (
Tetrahedron Lett
. 34, 3143 (1978)), and Ohtsuka et al. (
Nucleic Acids Res
. 10, 6553 (1982)) all disclose use of dimeric or multimeric synthons in a phosphotriester approach. Kumar and Poonian (
J. Org. Chem
. 49, 4905 (1984)) and Wolter et al. (
Nucleosides and Nucleotides
5, 65 (1986)) d
Bongle Nandkumar
Gonzalez Jose
Schwartz Warren E.
Tang Jin-Yan
Avecia Biotechnology Inc.
Hamilton Brook Smith & Reynolds P.C.
Houtteman Scott W.
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